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128 Forde
3. Methods
3.1. Production of Glutathione-Streamline Matrix
The following procedure is used to produce a glutathione-Streamline matrix
for use in the packed bed and expanded bed chromatography studies. The
method uses a bisoxirane to introduce oxirane (epoxy) groups to a hydroxylic
polymer adsorbent. An epoxy-activated adsorbent (Streamline) is used to
covalently immobilize a ligand (glutathione) containing amine or thiol groups.
1. Wash 30 ml of settled bed volume Streamline particles extensively in DI water
(see Note 3).
2. Remove excess water and resuspend particles in 23 ml of 0.6 M NaOH containing
45 mg sodium borohydride (see Note 4).
3. Add 23 ml of 1,4-butanediol diglycidyl ether (BDGE, MW 202.25, 95%) with
constant stirring (see Note 5). The matrix is activated by stirring for 24 h at
37°C. Stirring is performed using a rotary incubator (set at 100 rpm) as the use
of a magnetic stirrer may result in damage of the adsorbent.
4. The next day, wash the matrix extensively with water to remove excess reagent.
5. Remove excess water and resuspend the particles in 30 ml of 100 mM NaHCO 3 ,
pH 8.5 (see Note 6).
6. Remove the resuspension liquid, then add 30 ml of glutathione ligand solution:
0.5 mmol reduced glutathione/ml adsorbent, 100 mM NaHCO 3 , pH 8.5 (see
Note 7).
7. Stir the solution at 37°C for 24 h.
8. Wash the gel three times with PBS buffer to remove unreacted ligand.
9. Block excess active groups on the particles by adding 30 ml of 1 M ethanolamine
(pH 9) and stir at room temperature for 6 h.
10. Wash the particles with PBS and store as a 50% slurry at 4°C.
3.2. Ligand Density Measurement: Free Amine Groups
1. Create a ninhydrin reagent by dissolving ninhydrin in DI water to produce a
0.10 M solution (see Note 8).
2. Add 1 ml of reagent to 1 ml of a 50% slurry of adsorbent in DI water.
3. Incubate the solution on a rotary stirrer for 1hatroom temperature.
4. Centrifuge the sample briefly (max speed, 1 min) using a microcentrifuge in order
to settle out the adsorbent particles.
5. Measure the optical density at 564 nm (OD 564 nm ) of the supernatant and compare
the results to a calibration curve created using a serial dilution of pure reduced
glutathione in DI water (0.1 g/ml to 5×10 −6 g/ml is a good starting point).
3.3. Ligand Density Measurement: Free Thiol Groups
1. Dithiodipyridine is sparingly soluble in water. In order to produce the
dithiodipyridine reagent, add a known amount of dithiodipyridine to DI water and